The role of RhoA in corticogenesis

Böhringer, Christian (2012): The role of RhoA in corticogenesis. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Abstract

Insights into the developmental processes during which the brain forms from the neuroepithelium may provide a deeper understanding how the brain works. The Rho family of small GTPases is known for its many cell biological functions such as regulation of the cytoskeleton, gene expression, cell migration, adhesion, cell polarity and the cell cycle. All of these functions are of importance during the formation of the cerebral neocortex, which consists of the generation of its different cell types, their migration to their destination and their maturation to a functional network. These roles have been mostly established in vitro using dominant negative or constitutively active constructs. Since these approaches are often not entirely specific for single pathways, this work used the Cre/loxP system to genetically delete an individual member of the Rho family, RhoA, to examine its role following a loss-of-function approach. Specifically, we examined a mouse line where part of the RhoA gene has been deleted by means of the Emx1::Cre mouse line. This idea is based on previous experiences with the deletion of Cdc42 in the developing neocortex, which leads to a loss of apical progenitors. RhoA often works as a functional antagonist to Cdc42. Using immunofluorescence, we could detect a loss of RhoA at embryonic day 12 (E12) in Emx1::Cre-positive offspring carrying the floxed RhoA-construct in both alleles (cKO). At E14, we detected an increase in mitotic cells to 160% (±25%, p<0,05) that decreased to 140% (±10%, p<0,05) at E16. In addition, these mitoses were no longer restricted to their specific zones, but rather scattered throughout the developing cortex. This change did not coincide with a severely changed proportion of Pax6-pos. apical progenitors and Tbr2-pos. basal progenitors. Investigating the cellular architecture of the developing cortex, we observed a loss of the radial orientation of radial glial cells, likely due to the disruption of the apical band of adherens junctions, which is the first effect observed after loss of the protein, and the consequent formation of rosette like structures in the brain parenchyma. Despite the severe cortical malformations at embryonic stages, the mice get born and reach the age of weaning at no apparent difference from the Mendelian rate. These post-natal animals display a phenotype known as subcortical band heterotopia or "double-cortex". The phenotype is characterized by changes in the formation of the cortical layers. Between the characteristic six-layered structure of the cortex (homotopic cortex) and the ventricle, we found a second, unlayered neuronal structure embedded in the white matter (heterotopic cortex). By means of immunofluorescence and BrdU birthdating experiments, we observed that this structure consists of neurons of all layers and generated at all stages of neurogenesis, with late-born neurons of upper-layer identity being the majority. In addition, we found astrocytes and interneurons rather evenly distributed throughout both cortical structures. Finally, by using in-utero electroporation to delete RhoA in individual cells, we found out that the misplacements of neurons in the heterotopic cortex was not due to an inability of RhoA-neg. neurons to migrate. This lead us to the conclusion, that the neuronal misplacement is a secondary effect, which occurs due to the observed disruption of the radial glial structure. Looking for molecular pathways that may be at the start of these defects, we could observe a decrease of F-actin levels in RhoA-neg. progenitor cells in culture. Since F-actin stabilizes adherens junctions, RhoA's regulation of actin levels might indeed be at the origin "double-cortex" phenotype. Taken together, our data show an important role of RhoA in developing cortex. In addition they show, that defects in the radial glial scaffold are enough to induce the formation of a "double-cortex".

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: neurobiology, developmental biology, stem cells, radial glia
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 540 Chemie
• Fakultäten: Fakultät für Chemie und Pharmazie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 8. Mai 2012
• 1. Bericht­erstatter:in: Culmsee, Carsten